Clostridium strains, which produce toxin with neurotoxicity, have been found since 1890 up to present, and research into the properties of toxin produced by Clostridium strains has been conducted for 70 years (Schant, E. J. et al, Microbiol. Rev. 56; 80; 1992).
The genus Clostridium has more than 127 species, and is grouped according to their morphology and functions. Anaerobic and Gram-positive bacteria Clostridium botulinum produces a polypeptide neurotoxin that causes neural paralysis, which is referred to as botulism, in humans and animals. Spores of Clostridium botulinum are found in soil, and a large amount thereof can be germinated and cultured even in not fully processed foods. These can be causes of botulism.
Botulinum toxin is divided into seven types A through G according to their serological characteristics. Each toxin has about 150 kDa of toxin protein, and naturally comprises a complex with a variety of non-toxic proteins bound thereto. An intermediate complex (300 kDa) comprises toxin protein and non-toxic non-hemaglutinin protein, and a large complex (500 kDa) and a macro complex (900 kDa), each has a structure in which the intermediate complex is bound to hemaglutinin (Sugiyama, H., Microbiol. Rev., 44, 419; 1980). Such a non-toxic non-hemaglutinin protein is known to protect a toxin from a low pH of intestines, and various kind of protein hydrolytic enzymes (Sugiyama, H., Microbiol. Rev., 44, 419; 1980).
Botulinum toxin is synthesized in a cell, as a single-chain polypeptide, with a molecular weight of approximately 150 kDa, and then cleaved into two subunits at a position that is one-third the distance from an N-terminal by using protease in the cell or by using an artificial enzyme treatment such as trypsin: a light chain with a molecular weight of 50 kDa and a heavy chain with a molecular weight of 100 kDa. Such toxin cleaved into two subunits has highly increased toxicity in comparison with the single-chain polypeptide. The two subunits are bound to each other by a disulfide bond, and each subunit differently functions. That is, the heavy chain binds with a receptor of a target cell (FEMS Microbiol. Lett. 72, 243; 1990), and reacts with a bio-membrane at a low pH (pH 4.0) to form a channel (Mantecucco, C. et al, TIBS 18, 324; 1993). On the other hand, the light chain has pharmacological activities, and thus when the light chain is introduced into the cell by electroporation or enhanced permeability caused by a surfactant, the light chain interferes with secretion of a neuro-transmitter (Poulain, B. et al, Proc. Natl. Acad. Sci. USA. 85, 4090; 1988).
Botulinum toxin type A is the most lethal natural biological agent known to man. On a molar basis, Botulinum toxin type A is 1.8 billion times more lethal than diphtheria toxin, 600 million times more lethal than sodium cyanide, 30 million times more lethal than cobratoxin and 12 million times more lethal than cholera toxin (Singh, Critical Aspects of Bacterial protein Toxins, page 63-84 of natural toxins II, edited by B. R. Sigh et al., Plenum Press, New York (1976)).
Botulinum toxin inhibits exocytosis of acetylcholine from a cell at a cholinergic presynapse of a neuromuscular junction, thereby causing asthenia. Taking into account that in spite of the treatment of a very small amount of Botulinum toxin, toxicity is exhibited, it is considered that Botulinum toxin may have an enzyme activity (Simpson, L. L. et al, Ann. Rev. Pharmaeol. Toxicol. 26, 427; 1986). According to recent reports, Botulinum toxin has a metallopeptidase activity, and a substrate thereof is unit proteins of an exocytosis machinery complex, such as synaptobrevin, syntaxin, and synaptosomal associated protein of 25 kDa (SNAP 25). Each type of Botulinum toxin uses one of the three proteins as a substrate, and it is known that Botulinum toxin type B, D, F, and G cleave synaptobrevin at a specific site, Botulinum toxin type A and E cleave SNAP25 at a specific site, and Botulinum toxin type C cleaves syntaxin at a specific site (Binz, T. et al, J. Biol. Chem. 265, 9153; 1994).
WO06/096163 discloses that a seed culture and a main culture are performed under nitrogen, and the pH is adjusted from 5 to 5.5 in order to express Botulinum toxin protein. In addition, multi-step chromatography is performed, and washing and sterilization must be performed using reusable equipment and consumables. Such a system requires many labor and high investment costs, and high risk factors exist in the system in order to meet cGMP (current good manufacturing practice) requirements.